The invention relates to the field of real time sorting systems for rapid collection of data about objects or signals, arrangement of that data into a multidimensional space vector, characterization of the object based upon its signature from the multidimensional space vector and directing an output device to carry out a sort decision or other action to characterize the object or signal. A specific application of the system is in the field of cell sorting for biological research where a stream of dyed cells is passed through a laser beam, scatter and fluorescence data is collected and each cell's phenotype is established in real time by a computer comparison of the data After the phenotype is established, a sort decision is made and carried out.
There are many fields where real time sorting or characterization of rapidly occurring events is necessary. Those skilled in the art of real time sorting of objects based upon characterizing the objects using a multidimensional binary sort tree will appreciate the types of applications which are in need of a system such as the one disclosed herein.
In the field of biological research, it is useful to be able to sort large numbers of different cells into different phenotypes characterized by different properties. This process cannot be done by hand. Automated cell sorters were developed in the last 20 years to sort several thousand cells per second passing an inspection point in a stream of liquid. These machines used electrostatic droplet deflection technology originally developed for ink jet printers. Early versions of such sorters used laser beams shining on a stream of liquid carrying the cells of interest. The cells had been dyed with dyes which exhibited fluorescence. These cells would both absorb and scatter the laser light as they passed the beam. The dyes on the cells would be excited by the laser light and fluoresce, i.e., emit light in certain bands of wavelengths as the cells moved further in the stream.
This scattered and fluorescent light was collected by sensors and analyzed by analog circuitry. Because certain dyes were bound chemically to certain antibodies and because those antibodies would only bind to certain proteins on the surface of the cells of interest, each cell would have a unique phenotype expressed in terms of the light scattering and emission properties of the cell with its fluorescent tag. Such phenotypes could be used to make sorting decisions to enable sorting of the cells into physically separate collection containers. This was done in the prior art by generating a sort decision and using it to charge a drop containing the cell. Each drop would be charged with a charge having a polarity such that the drop could be deflected into the proper collection container when the drop passed through an electrical field between two electrically charged high voltage plates. These plates had a potential difference between them which created an electric field of a polarity to deflect the trajectory of the drop containing the cell having a particular phenotype into the corresponding collection container. Charging and formation of the drops was done by vibrating a nozzle the cells passed through. An electrode was placed in the stream of liquid. This electrode was coupled to the sorting circuitry and could be charged to any of a number of different states. The vibration of the nozzle caused the emerging liquid jet to be broken into thousands of individual, uniform droplets. These droplets were charged with whatever charge was on the electrode at the time the droplet broke from the jet.
The structure and operation of these early analog sorters is more fully described in U.S. Pat. No. 3,826,364 and in Chapter 19 of Volume 108 of the Methods in Enzymology series, by D. R. Parks and L. A. Herzenberg entitled, Fluorescence-Activated Cell Sorting: Theory, Experimental Optimization, and Applications in Lymphoid Cell Biology, Academic Press, Orlando, Fla. (1984). Another reference useful for understanding the background of and foundation technology for cell sorting, also called flow cytometry, is Chapter 29 of the Handbook of Experimental Immunology, 4th edition by D. R. Parks, L. L. Lanier & L. A. Herzenberg entitled Flow Cytometry and Fluorescence Activated Cell Sorting (FACS), Blackwell Scientific Publications, Oxford, England (1986). Machines to do such sorting are commercially available from Becton Dickinson as the FACS I, FACS II, FACS III, FACS IV, FACS 400, FACS 420, FACS 440 and FACSTAR. Some of these machines have been in use at Stanford University in the Department of Genetics for many years, and the details of their design are well known. Other companies also make flow cytometry machines available to the public. These publications and the knowledge embodied in these machines are hereby incorporated by reference.
The trend in flow cytometry is toward multiparameter measurements using multiple laser wavelengths exciting multiple dyes with differing emission spectra so as to enable more sophisticated and powerful analysis. In the prior art, people have generally been collecting only two or three dimensional data consisting of forward scatter and one or two wavelengths of fluorescence. This data was either analyzed using analog circuitry or digitized and used as indices in a look up table which stored sort decisions for various phenotypes characterized by various scatter and fluorescence parameters. The sorting decisions were then output into a buffer to await arrival of the droplet containing the cell to which each sort decision applied at the sorting apparatus of the machine. Output of sorting decisions from the buffer to the electronic circuitry that controlled the charging of the droplets was based upon a digitized index value for each cell. This index value was based upon the time of arrival of the cell in the area of the machine where it was excited by the laser beam.
The problem with this approach was that the sophistication of the phenotype sorting decision was limited, and the sorting decision was somewhat inflexible in the analog case since new sorting decision processes had to be implemented in new analog hardware.
These limitations led people to search for more sophisticated and flexible systems. One step was to increase the number of lasers and dyes used so that additional properties could be measured for each cell, resulting in additional dimensions in the data vector for each cell. These developments are described in a paper by D. R. Parks, R. R. Hardy and L. A. Herzenberg from the Stanford University Department of Genetics in the School of Medicine, entitled "Three-Color Immunofluorescence Analysis of Mouse B-Lymphocyte Subpopulations" published in Cytometry, Vol. 5, p. 159 (1984). With such data, much more sophistication of analysis was possible and more phenotypes could be distinguished. This paper is hereby incorporated by reference.
The ability to collect multiple parameters in multidimensional space, i.e., space having more than two dimensions has led to severe complications in trying to use prior art analog and digital systems to make sort decisions on this type of data in real time and with sufficient flexibility to be able to easily change the methodology of the sorting decision. Analog systems must be extremely complex to implement this type of sort decision and can implement only one type of analysis for a given design of the analog sort decision hardware. If a different type of analysis is desired, new sort decision hardware has to be built.
Prior art digital flow cytometry systems using lookup tables cannot handle multidimensional space data having three or more dimensions. Array look up algorithms do not extend well to multidimensional space. Such array look up flow cytometry systems require prohibitive amounts of memory which memory demand increases exponentially when more dimensions are to be considered in sort decisions.
Further, all prior art devices known to the inventors require a cell to pass in front of both lasers before the peak detect and sampling circuitry can be released to deal with another cell. This need for each cell to pass in front of both lasers before it could be classified caused a blind spot in prior art devices during which no other events could be detected and no other cell could be classified. This blind spot had a duration equal to the travel time for the cell to pass in front of both lasers.
Further, there is no prior art sorter which can receive compensation signals from more than one channel, sum the compensation signals and use the composite signal for compensation purposes.
Thus a need has arisen for a cell sorting system which can be fast enough to operate in real time on multidimensional space vectors and flexible enough to allow differing analysis and decision making processes to be implemented easily without difficult, time consuming and expensive modification of the apparatus.
As far as applicants know, there is no prior art system wherein sorting decisions can be made without the need for a cell to pass in front of both lasers. Also, there is no prior art cell sorter system wherein the compensation circuitry for each channel can receive compensation signals from more than one other channel to eliminate cross talk resulting from dye emissions from more than one other channel falling within the passband of the channel being compensated.